Classification of Andrographis paniculata extracts by solvent extraction using HPLC fingerprint and chemometric analysis.

OBJECTIVE: Andrographis paniculata, widely used as an antidiabetic in Indonesian traditional medicines (jamu), contains chemical compounds whose concentration is related to its therapeutic effects. The concentration of solvents used for extraction will also affect the number of compounds extracted. Therefore, a quality control method is needed to ensure consistency in quantifying these compounds in A. paniculata to improve its therapeutic application. High-performance liquid chromatography fingerprint analysis combined with chemometrics was used to evaluate extracts from different solvent extraction treatments. The content of andrographolide, the main bioactive compound in A. paniculata, and the level of α-glucosidase inhibition activity, an indicator of its antidiabetic activity, were also determined. RESULTS: Fingerprint chromatograms of A. paniculata extracts from different treatments exhibited a similar pattern with several peaks in common, only differing in area and intensity value. The A. paniculata extracts were classified using HPLC fingerprint and principal component analysis to allow grouping according to their respective solvent extraction treatments. The highest andrographolide content and α-glucosidase inhibition activity occurred in the 50% ethanol extract and the lowest in the water extract. HPLC fingerprint analysis could be used for identifying A. paniculata extracts based on solvent extraction, thus improving quality control for their therapeutic application.

Effect of Zingiber zerumbet essential oils and zerumbone inhalation on body weight of Sprague Dawley rat.

Zingiber zerumbet contained the typically essential oils. The research aims to evaluate the effect Z. zerumbet essential oil and zerumbone inhlation on rats body weight, food consumption, parasympathetic nerve activity and brown adipose tissue temperature. The essential oils of Z. zerumbet was isolated from the rhizome of Z. zerumbet. The component in the oil and zerumbone structure was determined by gas chromatography-mass spectroscopy. The structure of zerumbone crystal was determined by nuclear magnetic resonance spectroscopy. The Sprague dawley male adult rats were divided into 4 groups namely Normal Diet (ND) group, High Fat Diet (HFD) group, HFD inhaled Z. zerumbet essential oils group and HFD inhaled zerumbone group. The results showed that inhalation of Z. zerumbet essential oils and zerumbone increased the food consumption as well as increased the body weight. The increasing body weight of rats which inhaled Z. zerumbet essential oils and zerumbone is by decreasing the sympathetic nerve activity. In conclusion, inhaling Z. zerumbet essential oils and zerumbone as the major component of the oils increased the weight gain.

Experimental infection of Macaca nemestrina with a Toronto Norwalk-like virus of epidemic viral gastroenteritis.

Norwalk virus (NV) and Norwalk-like viruses (NLVs) are common etiologic agents of viral gastroenteritis. Viral gastroenteritis is a common disease that is highly transmissible, spreading rapidly through families, institutions, and communities. Because methods for in vitro cultivation of Norwalk etiologic agents are not available, information regarding this syndrome has come largely from studies in human volunteers. Sequential passaging of an NLV through an immunoincompetent newborn pigtail macaque (Macaca nemestrina) may allow for the adaptation of a human NLV to a primate host, thus providing an animal model for investigating this disease. A fecal filtrate of human origin containing NLV, Toronto virus P2-A, was obtained from a patient during an epidemic of viral gastroenteritis. The filtrate was administered via nasogastric tube to three newborn pigtailed macaques. Clinical illness, which was characterized by diarrhea, dehydration, and vomiting, occurred in three monkeys. Reverse transcription-polymerase chain reaction (RT-PCR) and oligonucleotide probe analysis of RNA extracted from the stool samples following infection revealed viral RNA in all inoculated monkeys. Infection was also transmitted experimentally by feeding two additional newborn macaques a fecal filtrate prepared from the three previously infected animals. Detection of viral RNA in the stools of animals that received the fecal filtrate indicates that viral replication occurred in association with clinical illness. The susceptibility of Macaca nemestrina to infection with a Norwalk-like agent will facilitate the study of the mechanisms of the pathogenesis of NLV. This system may also have the potential to serve as a vaccine test model for human epidemic viral gastroenteritis.

CpG DNA overcomes hyporesponsiveness to hepatitis B vaccine in orangutans.

Oligonucleotides containing immunostimulatory CpG motifs (CpG ODN) have been shown to be potent Th1-type adjuvants for augmenting antigen-specific responses in mice against hepatitis B surface antigen (HBsAg). The hepatitis B virus (HBV) infects only humans and great apes and appears to exist among wild chimpanzees and orangutans. An outbreak of HBV among orangutans being rehabilitated for re-introduction to the jungle caused the death of several animals. A prophylactic vaccination program revealed that orangutans are quite hypo-responsive to a current commercial vaccine compared to results obtained previously in humans and chimpanzees. Addition of CpG ODN to hepatitis B vaccine greatly increased the seroconversion rate and the titers of antibody against HBsAg (anti-HBs). This is the first demonstration of CpG DNA in a great ape and the results have important implications for the vaccination of humans against HBV and other diseases.

Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo.

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are detected, like bacterial or viral DNA, as a danger signal by the vertebrate immune system. CpG ODN synthesized with a nuclease-resistant phosphorothioate backbone have been shown to be potent Th1-directed adjuvants in mice, but these motifs have been relatively inactive on primate leukocytes in vitro. Moreover, in vitro assays that predict in vivo adjuvant activity for primates have not been reported. In the present study we tested a panel of CpG ODN for their in vitro and in vivo immune effects in mice and identified in vitro activation of B and NK cells as excellent predictors of in vivo adjuvant activity. Therefore, we tested >250 phosphorothioate ODN for their capacity to stimulate proliferation and CD86 expression of human B cells and to induce lytic activity and CD69 expression of human NK cells. These studies revealed that the sequence, number, and spacing of individual CpG motifs contribute to the immunostimulatory activity of a CpG phosphorothioate ODN. An ODN with a TpC dinucleotide at the 5' end followed by three 6 mer CpG motifs (5'-GTCGTT-3') separated by TpT dinucleotides consistently showed the highest activity for human, chimpanzee, and rhesus monkey leukocytes. Chimpanzees or monkeys vaccinated once against hepatitis B with this CpG ODN adjuvant developed 15 times higher anti-hepatitis B Ab titers than those receiving vaccine alone. In conclusion, we report an optimal human CpG motif for phosphorothioate ODN that is a candidate human vaccine adjuvant.